The Ras family of small guanine-nucleotide binding proteins plays a pivotal role in many intracellular signal transduction pathways, including those which regulate cellular growth and differentiation and those which contribute to cell activation (Bourne et al., Nature 348:125-132 (1990); Marshall, FASEB J. 9:1311-1318 (1995)). Moreover, many different receptors expressed on the surface of diverse cell types can result in the activation of signal transduction pathways that are importantly influenced by Ras, and these pathways in turn determine whether, and to what extent, these cells respond to such cell surface receptor-dependent activation by proliferating, differentiating (i.e., developing new functional characteristics), and/or expressing specific functions (FIG. 1).
Depending on the circumstances, these xe2x80x9cdown-streamxe2x80x9d consequences of the activation of Ras-dependent signal transduction pathways can have either adaptive (physiological) or maladaptive (pathological) consequences. For example, controlled Ras-dependent cellular proliferation is required for wound healing, whereas poorly regulated Ras-dependent cellular proliferation can result in the development of cancer and other neoplasms. Similarly, appropriate Ras-dependent cell secretion of histamine, serotonin, cytokines and other mediators can be important for host defense against parasites and other pathogens, whereas the inappropriate activation of these same pathways, for example, by a reaction to a bee-sting in patients who are allergic to components of bee venom, can lead to fatal anaphylaxis. Thus, Ras represents a major regulator of many of the most fundamental biological processes involved in both health and disease.
The mechanism by which Ras regulates such processes, through interactions with other intracellular molecules, is quite complex. Ras proteins are membrane-associated proteins that cycle between an active GTP-bound form and an inactive GDP-bound form. As illustrated in FIG. 1, evidence is accumulating for the existence of many different classes of positive or negative regulators of Ras and positive or negative Ras signaling effectors, all of which, by definition, are thought to interact directly with the active GTP-bound form of Ras to influence cellular signaling for growth, differentiation and expression of function (Boguski and McCormick, Nature 366:643-654 (1993); Marshall, FASEB J. 9:1311-1318 (1995); Marshall, Curr. Opin. Cell Biol 8:197-204 (1996)).
For example, some of the best-characterized Ras regulators include the GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (Boguski and McCormick, Nature 366:643-654 (1993)). The GAPs represent a family of Ras-binding proteins which stimulate the intrinsic rate of Ras GTP hydrolysis and thus negatively regulate the Ras-induced signaling by accelerating the conversion of active GTP-bound form of Ras to the inactive GDP-bound form. Recent studies have identified several GAPs specific for Ras proteins, which include p120-Ras GAP, neurofibromin (the protein encoded by the neurofibromatosis type 1 (NF1) gene), Gap1, Ral-GDS, Rsbs 1, 2, and 4, Rin1, MEKK-1, and phosphatidylinositol-3-OH kinase (P13K) (Boguski and McCormick, Nature 366:643-654 (1993)).
In contrast to Ras regulators, which function primarily by influencing the amount of Ras which is in the GTP-bound active, as opposed to the GDP-bound inactive, form, Ras effectors are thought to influence the ability of active, GTP-bound Ras to initiate signaling (FIG. 1). In the case of many Ras-interacting proteins which can influence the intensity of Ras-dependent signaling, it is not yet clear to what extent they function as effectors as opposed to regulators; such proteins can therefore be called Ras regulators/effectors (Boguski and McCormick, Nature 366:643-54 (1993); Han and Colicelli, Mol. Cell. Biol. 15:1318-1323 (1995); Marshall, FASEB J. 9:1311-1318 (1995); Marshall, Curr. Opin. Cell. Biol. 8:197-204 (1996)).
Ras is important in critical cell signaling events in many cell types. For example, mast cells are important effector cells in IgE-dependent immune responses and allergic diseases (Galli, New Engl. J. Med. 328:257-265 (1993)), and mast cells also contribute to host defense against parasites and bacteria (Echtenacher et al., Nature 381:75-77 (1996); Malaviya et al., Nature 381:77-80 (1996); Galli and Wershil, Nature 381:21-22 (1996)). Mast cells reside in virtually all vascularized tissues and express on their surface the high affinity receptor for IgE (Fcxcex5RI). Aggregation of Fcxcex5RI in mast cells by the interaction of receptor-bound IgE with specific multivalent antigen triggers the functional activation of mast cells, which results in the release of a spectrum of biologically active mediators (Ravetch and Kinet, Ann. Rev. Immumol. 9:457-492 (1991); Galli, New Engl. J. Med. 328:257-265 (1993); Beaven and Metzger, Immunol. Today 14:222-226 (1993)). Thus, mast cells activated by Fcxcex5RI-dependent mechanisms undergo degranulation, resulting in the release of preformed mediators, such as serotonin (5-HT) and/or histamine, the metabolism of arachidonic acid, leading to the release of newly synthesized lipid mediators, and the transcription, translation and secretion of several cytokines (Gordon et al., Immunol. Today 11:458-464 (1990); Galli, New Engl. J. Med. 328:257-265 (1993); Paul et al., Adv. Immunol. 53:1-29 (1993)).
Knowledge of the signaling pathways which result in the Fcxcex5RI-dependent secretion of mast cell mediators is increasing. In mast cells and basophils, a type of circulating leukocyte which shares many biochemical and functional characteristics with mast cells (Galli, New Engl. J. Med. 328:257-265 (1993)), the Fcxcex5RI receptor is a tetrameric complex comprised of a single 45 kDa xcex1 chain, which binds the Fc portion of IgE, a single 30 kDa xcex2 chain, and a homodimer of two 10 kDa xcex3 chains (Ravetch and Kinet, Ann. Rev. Immunol. 9:457-492 (1991); Beaven and Metzger, Immunol. Today 14:222-226 (1993)). The xcex2 and xcex3 chains contain immunoreceptor tyrosine-based activation motifs (ITAM) which couple the receptor to the src family of protein tyrosine kinases (PTK) p561yn and p72syk (Beaven and Baumgartner, Curr. Opin. Immumol. 8:766-772 (1996)). The Fcxcex5RI-dependent activation of these PTKs in turn activates various downstream effector pathways, including those involving PLCxcex31 and the MAP kinase pathway (Beaven and Baumgartner, Curr. Opin. Immumol. 8:766-772 (1996)).
Recent studies have shown that the crosslinking of Fcxcex5RI in mast cells by IgE and specific antigen also results in the activation of Ras and of the associated Shc-Grb2-Sos pathway, which precedes Ras activation, and that the activation of this pathway is dependent on Syk (Jabril-Cuenod et al., J. Biol. Chem. 271:16268-16272 (1996)). These results suggest that in Fcxcex5RI-activated mast cells, as in T cells and B cells (the major types of lymphocytes responsible for cellular and humoral immunity) which have been activated via the T cell receptor or B cell receptor, respectively, the Shc-Grb2-Sos pathway can activate the MAP kinase pathway via the activation of Ras.
An effector pathway of Ras mediated by the Raf-1/Erk-activating kinases (MEKs)/Erk-MAP kinases cascade has been well-characterized in numerous systems (Treisman, Curr. Opin. Cell Biol. 8:205-215 (1996)), and this Ras-mediated pathway is important in the mast cell activation and mediator secretion that is induced by IgE- and antigen-dependent aggregation of Fcxcex5RI in these cells (Tsai et al., Eur. J. Immumol. 23:3286-3291 (1993); Offermanns et al., J. Immunol. 152:250-261 (1994); Hirasawa et al., J. Immunol. 154:5391-5402 (1995)). In addition, recent studies with rat RBL2H3 mast cells have shown that the Fcxcex5RI induction of Ras activation leads to transcriptional activation mediated by the transcription factors Elk-1 and the nuclear factor of activated T cells (NFAT) (Turner and Cantrell, J. Exp. Med. 185:43-53 (1997)). These Ras-dependent signaling pathways in mast cells appear to be complex. Thus, activation of the Raf-1/MEK/Erk cascade appears to be necessary and sufficient for the activation of Elk-1 activity in mast cells. However, the induction of NFAT by Fcxcex5RI in mast cells is also mediated in part by Rac-1, which is a putative Ras effector and a member of the Rho family of GTP binding proteins (Turner and Cantrell, J. Exp. Med. 185:43-53 (1997)). Thus, the specific signaling pathways involved in Fcxcex5RI-mediated mast cell activation are still unclear.
As described herein, mRNA differential display was used to identify the cDNA for a novel gene, rin2 (also called Rabex-5). As also described herein, expression of rin2 was shown to be rapidly increased in mouse mast cells activated through Fcxcex5RI. Transfection of mouse mast cells with an antisense rin2 expression vector, which resulted in antisense inhibition of Rin2 expression, potentiated the Ras-mediated intracellular signaling responses that were induced by Fcxcex5RI aggregation, such as the induction of c-fos expression and the activation of Erk-MAP kinase, JNK and p38 MAP kinase activity. In addition, antisense inhibition of rin2 expression in mouse mast cells significantly enhanced the amounts of preformed mediator (serotonin) and cytokine (IL-6) released from these cells upon Fcxcex5RI-dependent stimulation, suggesting that Rin2 exerts its effects by down-regulating the functional responses elicited by Fcxcex5RI aggregation in mast cells. These results support the role of Rin2 as a novel negative regulator/effector of Ras in mast cells and indicate that Rin2 interacts directly with the mammalian H-Ras protein.
Moreover, as also described herein, expression of rin2 is also increased in mast cells stimulated via activation of their major growth factor receptor (i.e., c-kit), in PC12 adrenal pheochromocytoma cells activated via the receptor for nerve growth factor (NGF) (i.e., TrkA), and in T cells activated via the T cell receptor (TCR). These findings support the role of Rin2 as a novel general negative regulator/effector of Ras and Ras-dependent signaling pathways in diverse cell types which have been activated via distinct cell surface receptors (FIG. 2). In addition, the enhancement or inhibition of Rin2 expression or function, in certain settings, resulting in the reduction or enhancement, respectively, of Ras-dependent signaling in these settings, may have benefit in diverse clinical problems, some examples of which are shown in FIG. 2.
As also described herein, the SY-A (Rin2) clone has been used as a probe to screen a HT-29 human adenocarcinoma cDNA library. Two overlapping cDNA clones (HRIN2-1B1 (1.3 kb) and HRIN2-2-B1 (2.9 kb)) have been identified which are highly homologous to that of the mouse Rin2 clone and which together represent the full-length human Rin2 (Rabex-5) cDNA sequence. The orientation of these overlapping clones is shown in FIG. 9. The nucleotide sequence of each clone is shown in FIGS. 10A-B and 11A-C, respectively.
Accordingly, this invention pertains to isolated Rin2 protein, which down-regulates the functional responses elicited by Ras-dependent signaling pathways. In one embodiment, Rin2 down-regulates the functional responses elicited by Fcxcex5RI aggregation in mast cells (e.g., mammalian mast cells), including down-regulating the amounts of preformed mediator (serotonin) and cytokine (IL-6) released from these cells upon Fcxcex5RI-dependent stimulation. In another embodiment, Rin2 down-regulates functional responses in mast cells stimulated via activation of their major growth factor receptor (i.e., c-kit). In another embodiment, Rin2 inhibits cell proliferation in PC12 adrenal pheochromocytoma cells stimulated through the nerve growth factor receptor (i.e., TrkA). In a further embodiment, Rin2 inhibits functional responses in T cells activated through the T cell receptor.
In one embodiment, isolated Rin2 protein has the amino acid sequence of SEQ ID NO: 2. In one embodiment, the Rin2 protein is a portion of SEQ ID NO: 2 which is sufficient for Rin2 activity, e.g., binding or enzymatic activity. Also included within the meaning of Rin2 protein or polypeptide are polypeptides which are less than the amino acid sequence of SEQ ID NO: 2 and have Rin2 activity, as well as proteins whose amino acid sequence is substantially similar to that of SEQ ID NO: 2 and have Rin2 activity.
In another embodiment, Rin2 protein is a derivative possessing substantial sequence identity with the endogenous Rin2 protein. In particular embodiments, the Rin2 protein is purified to homogeneity or is substantially free of other proteins.
The invention also pertains to an isolated nucleic acid molecule or nucleotide sequence (rin2) which encodes Rin2 protein or polypeptide, or a portion of SEQ ID NO: 2 which is sufficient for Rin2 activity. In one embodiment, the encoded Rin2 protein is a derivative possessing substantial sequence identity with the endogenous Rin2 protein. In a particular embodiment, the isolated nucleic acid molecule encodes Rin2 protein with the same amino acid sequence as endogenous Rin2 protein. In another embodiment, the isolated nucleic acid molecule has the same nucleotide sequence as the endogenous gene encoding Rin2 protein. In one embodiment, the isolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1. In another embodiment, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 12 and/or the nucleotide sequence of SEQ ID NO: 13. In a further embodiment, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 14 and/or the nucleotide sequence of SEQ ID NO: 15. In one embodiment the isolated rin2 nucleic acid molecule comprises the nucleotide sequence of one or more of SEQ ID NOS: 12, 13, 14 and 15.
The invention also relates to DNA constructs comprising the nucleic acid molecules described herein, operatively linked to a regulatory sequence, and to recombinant host cells, such as bacterial cells, fungal cells, plant cells, insect cells and mammalian cells, comprising the nucleic acid molecules described herein operatively linked to a regulatory sequence. The invention also relates to a method for preparing a Rin2 polypeptide, comprising culturing a recombinant host cell described herein.
The invention also pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to Rin2 protein, or a portion of SEQ ID NO: 2 which is sufficient for Rin2 activity; in a particular embodiment, the antibody is a monoclonal antibody. The invention also relates to a method for assaying the presence of Rin2 protein in a cell, e.g., in a tissue sample, comprising contacting said cell with an antibody which specifically binds to Rin2 protein.
The present invention also relates to an assay for identifying agents which alter the activity of Rin2 protein. For example, a cell population, e.g., a mast cell population, containing Rin2 protein can be activated with a stimulus which activates at least one Ras-dependent pathway in the cell in the presence of an agent to be tested, and the level of Rin2 activity can be assessed.
The invention further relates to methods of inhibiting the functional responses (e.g., proliferation, functional activation) elicited by activation of Ras-dependent signaling pathways in cells, comprising contacting the cells with an agent which enhances or mimics the activity of Rin2 protein. The invention also relates to a method of treating a mammal in need thereof to inhibit functional responses elicited by activation of Ras-dependent signaling pathways, comprising administering to a mammal an agent which enhances or mimics the activity of native Rin2 protein.
The invention further relates to methods of inhibiting the functional responses elicited by Fcxcex5RI aggregation, particularly in mast cells, comprising contacting a mast cell population with an agent which enhances or mimics the activity of native Rin2 protein. The invention relates to a method of treating a mammal in need thereof to inhibit the functional response elicited by Fcxcex5RI aggregation, comprising administering to a mammal an agent which enhances the activity of native Rin2 protein. For example, the invention relates to methods of inhibiting IgE and antigen-dependent release of mediators from mast cells. Such methods can be used to inhibit mediator release from mast cells and other effector cells that express Fcxcex5RI, such as basophils, monocytes/macrophages, dendritic cells, Langerhans"" cells and eosinophils, thereby ameliorating disorders such as asthma and allergic diseases (e.g., hay fever and atopic eczema).
The invention further relates to methods of enhancing functional responses (e.g., proliferation, functional activation) elicited by activation of Ras-dependent signaling pathways in cells, comprising contacting the cells with an agent which inhibits the activity of Rin2 protein. The invention also relates to a method of treating a mammal in need thereof to enhance functional responses elicited by activation of Ras-dependent signaling pathways, comprising administering to a mammal an agent which inhibits the activity of Rin2 protein.